Perspective Duggan, Vazvaei & Jenkins Key terms
Immunoaffinity purification: Also termed immunoaffinity capture, in PrD-LCMS, this is the purification of the analyte protein from the biological matrix via an immunoaffinity technique using specific antibody or ligand-binding reagent. This term may also include immunopurification of the analyte prior to digestion or immunopurification of the surrogate peptide after digestion (SISCAPA).
PrD-LCMS: Specific abbreviation signifying the bioanalytical quantification of proteins via LC–MS/MS by monitoring of the SRM signal of a surrogate peptide after proteolytic digestion.
Surrogate peptide: Selected peptide that is specific to the target protein analyte, in the context of the assay and the intended sample matrix, and quantified in lieu of the target protein analyte in an LC–MS/MS assay. This peptide has also been named the target or signature peptide.
regulatory guidances [8,9] nor formally proposed in the literature, the recommendations provided in this White Paper [11] are supported by a recent European Bioanalysis Forum editorial on this subject [12]. These authors make the points that use of LC–MS(/MS) for large molecules is much more complex than for small molecules, analyzing peptides and proteins is a devel- oping science, and there is no compelling safety or PK need at this time to raise the standards over the cur- rently accepted 4–6–20 criteria used for LBA-based peptide/protein assays.
Validation parameters & acceptance criteria for LC–MS protein methods Some of the suggested validation requirements for PrD-LCMS [11] are summarized in Table 1 below, where they are also compared with existing LBA criteria. The consensus report recommends the well-known LBA 4–6–20 rules for precision and accuracy, including precision and accuracy for dilutional integrity/linearity samples [8,9]. That is, the acceptance criteria for stan- dards and QCs are that they should be within ±20% of the nominal concentration with reproducibility for multiple replicates of within 20% CV. These criteria are expanded to 25% accuracy and precision for QCs and standards prepared at the level of the LLOQ. The in-run ‘4–6’ criteria would require that four of six QCs (2/3) and 50% at each concentration level are required to meet acceptance criteria for run acceptance. A mini- mum of six calibration standards are deemed necessary to demonstrate the goodness of the fit with no more than 25% of the standards eliminated from the curve.
Selectivity/specificity The terms selectivity and specificity are often used interchangeably in bioanalysis, particularly with small molecule chromatographic methods. However, large
1390 Bioanalysis (2015) 7(11)
molecule hybrid methods apply a combination of LBA and chromatographic experimental procedures, and it is therefore important to take a closer look at the concepts of selectivity and specificity as they relate to large molecules. For LBAs, the EMA guidance [8] defines selectivity as the ability to measure the analyte of interest in the presence of ‘unrelated compounds’ in the matrix. Whereas specificity is generally related to the concept of cross-reactivity and defined as the ability (of critical affinity reagents) to bind solely to the analyte of interest in the presence of structurally ‘related compounds.’ When using affinity capture pro- cedures in hybrid LC–MS methods, it is desirable to keep these distinctions in mind and apply the appro- priate validation experiments to demonstrate freedom from interference. In the context of protein bioanalysis by LC–MS/
MS, we use the terms selectivity and specificity as a single combined concept, and we discuss the required validation experiments; therefore, we will use the term selectivity here as equivalent to ‘selectivity/specificity.’ Selectivity validation considerations may vary with the assay format (i.e., if an affinity capture step is used) and availability of potential interferents (e.g., endog- enous analogs, concomitant medications, isoforms, anti-drug antibodies [ADA], soluble targets and catab- olites). Generally, selectivity should be both evaluated during method development and confirmed during the validation exercise; however, specific interferents, nota- bly those contained in study samples, may not be avail- able during method validation. Therefore, selectivity should be considered as a continuing exercise, and it should often be evaluated further when study samples become available. This is particularly true for special patient populations or for the assessment of catabo- lites, ADAs and other possible interferents that may be found in incurred samples. As is done for small molecule chromatographic
assays, a minimum of six lots of blank matrix should be fortified with the analyte at the LLOQ level for selec- tivity evaluation. The analysis also includes extracted blanks, blanks with IS and LLOQ samples that were prepared in the matrix used for general validation. The response of the LLOQ sample should be at least five- times higher when compared with that of the blank sample. In addition, there should be no significant interference with the IS in the blank matrix (response in blanks ≤5% of the IS response). Moreover, the back calculated value for at least 80% of the LLOQ samples from the individual lots should be within 25% of the nominal value. For clinical studies, additional lots to test special patient population or hemolyzed and lipe- mic lots may be included, if study samples are expected to have those characteristics.
future science group
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